Method for preparing deuterated diphenylurea

ABSTRACT

Disclosed are an intermediate compound of N-(1,1,1-trideuterated methyl)phthalimide, and its use in the preparation of deuterated diphenylurea derivative.

FIELD OF INVENTION

This invention relates to the field of chemical synthesis, andparticularly relates to some intermediates for preparing deuterateddiphenylurea and the synthetic methods and the use thereof.

BACKGROUND OF INVENTION

The ω-diphenylurea derivatives are known as the compounds with c-RAFkinase inhibition activity. For example, W02000/042012 disclosed a classof co-carboxyl-aryl-substituted diphenylurea and the use thereof fortreating cancer and related diseases.

Initially, ω-diphenylurea compounds, such as Sorafenib, were firstlyfound as the inhibitor of c-RAF kinases. The other studies had shownthat they could also inhibit the MEK and ERK signal transductionpathways and activities of tyrosine kinases including vascularendothelial growth factor receptor-2 (VEGFR-2), vascular endothelialgrowth factor receptor-3 (VEGFR-3), and platelet-derived growth factorreceptor-β (PDGFR-β) (Curr Pharm Des 2002, 8, 2255-2257). Therefore,they were called multi-kinase inhibitors that resulted in dualanti-tumor effects.

Sorafenib (trade name Nexavar), a novel oral multi-kinase inhibitor, wasdeveloped by Bayer and Onyx. In December 2005, based on its outstandingperformance in phase III clinical trials for advanced renal cellcarcinoma, Sorafenib was approved by FDA for treating advanced renalcell carcinoma, and marketed in China in November 2006. However,Sorafenib has various side-effects, such as hypertension, weight loss,rash and so on.

However, the current procedure for preparing deuterated diphenylureacompounds is immature, and has some disadvantages, such as high-cost,low-yield, low-purity or difficulties in separation and so on.Therefore, novel and highly efficient procedures for preparingdiphenylurea compounds are needed to be developed.

SUMMARY OF INVENTION The subject of the invention is to provide highlyefficient procedures for preparing highly pure diphenylurea compoundsand the intermediates therein.

In the first aspect, the invention provides a method for preparing2-(methyl-d₃)isoindoline-1,3-dione, comprising:

(a) in an inert solvent, reacting a alkali metal salt of phthalimidewith compound A,

wherein, Z is CH₃, O—CD₃ or

wherein R is methyl, nitro or halogen (F, Cl or Br),

to form 2-(methyl-d₃)isoindoline-1,3-dione:

In one embodiment, said inert solvent includes N,N-dimethylformamide(DMF), N,N-dimethylacetamide (DMA), dimethylsulfoxide (DMSO),N-methylpyrrolidone (NMP), tetrahydrofuran, 1,4-dioxane, or thecombination thereof

In one embodiment, in step (a), the reaction temperature is -10° C. toreflux temperature; preferably is −4° C. to 100° C.; and more preferablyis 20-80° C.

In one embodiment, the reaction time is 0.1-24 hours; preferably is0.3-5 hours; and more preferably is 0.5-2 hours.

In one embodiment, in step (a), said alkali metal salt of phthalimideincludes (but is not limited to): potassium phthalimide, sodiumphthalimide, lithium phthalimide, or the combination thereof.

In one embodiment, in step (a), said compound A includes: methyl-d₃4-methyl-benzenesulfonate, methyl-d₃ 3-nitrobenzenesulfonate, ormethyl-d₃ 4-nitrobenzenesulfonate.

In one embodiment, prior to step (a) of said method it further includesstep (a1):

under a basic condition and in an inert solvent, reacting deuteratedmethanol with tosyl chloride to form methyl-d₃4-methyl-benzenesulfonate.

In one embodiment, in step (a1), said inert solvent includes water,tetrahydrofuran, or the mixture thereof.

In the second aspect, the invention provides a method for preparing asalt of (methyl-d₃)amine, comprising:

in an inert solvent or an aqueous solvent, reacting2-(methyl-d₃)isoindoline-1,3-dione with an acid to form a salt of(methyl-d₃)amine, wherein said acid includes: hydrochloric acid,sulfuric acid, hydrobromic acid, trifluoroacetic acid, or thecombination thereof.

In one embodiment, said reaction temperature is 30 to reflux temperature(such as 120° C.), preferably 40-110° C.

In one embodiment, said reaction time is 0.5-48 hours, preferably is1-36 hours, and more preferably is 2-24 hours.

In the third aspect, the invention provides an intermediate used forpreparing deuterated diphenylurea, wherein the intermediate is2-(methyl-d₃)isoindoline-1,3-dione.

In one embodiment, the intermediate is used for preparing deuterateddiphenylurea or as the raw material for preparing deuterateddiphenylurea.

In one embodiment, said deuterated diphenylurea is compound I:

wherein,

X is N or N⁺—O⁻;

R¹ is halogen (such as F, Cl or Br), one or more deuterium-substitutedor perdeuterated C1-C4 alkyl;

R² is non-deuterated C1-C4 alkyl, one or more deuterium-substituted orperdeuterated C1-C4 alkyl, or partly or totally halogen-substitutedC1-C4 alkyl;

each of R³, R⁴, R⁵, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ is independentlyhydrogen deuterium or halogen (such as F, Cl or Br);

R⁶ is hydrogen, deuterium, or one or more deuterium-substituted orperdeuterated C1-C4 alkyl;

R⁷ is hydrogen, deuterium, or one or more deuterium-substituted orperdeuterated C1-C4 alkyl; and

provided that at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³ or R¹⁴ is deuterated or is deuterium.

More preferably, said deuterated diphenylurea is selected from

4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-N-(methyl-d₃)picolinamide(CM4307);

4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-N-(methyl-d₃)picolinamidep-toluenesulfonate (CM4307.TsOH);

4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluorophenoxy)-N-(methyl-d₃)picolinamide(CM4309);

4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluorophenoxyl)-N-(methyl-d₃)picolinamidep-toluenesulfonate (CM4309.TsOH);

It should be understood that in the present invention, any of thetechnical features specifically described above and below (such as inthe Examples) can be combined with each other, thereby constituting newor preferred technical solutions that are not described one by one inthe specification.

DESCRIPTION OF FIGURES

FIG. 1 shows the curves of drug concentration (ng/ml) in plasma afteroral administration of 3 mg/kg of the control compound CM4306 to themale SD rats.

FIG. 2 shows the curves of drug concentration (ng/ml) in plasma afteroral administration 3 mg/kg of the compound CM4307 of the invention tothe male SD rats.

FIG. 3 shows the curves of inhibition efficacy of CM4306 and CM4307 innude mice xenograft model inoculated with human liver cancer cellSMMC-7721. In this figure, “treatment” means that the treating periodwas 14 days, followed by the observation period after administration wasstopped. The five days before treatment was the period for preparinganimal models.

DETAILED DESCRIPTION OF INVENTION

After studies, the inventors unexpectedly discovered that, compared withthe un-deuterated compound, the deuterated ω-diphenylurea of theinvention and the pharmaceutically acceptable salts thereof possessedbetter pharmacokinetic and/or pharmacodynamic properties. Therefore,they were much more suitable as raf kinase inhibitors for preparingmedicaments to treat cancer and the relevant diseases.

Furthermore, the inventors also discovered that diphenylurea compoundcan be more efficiently and easily prepared by using the newintermediate 2-(methyl-d₃)isoindoline-1,3-dione. Based on thisdiscovery, the inventors completed the present invention.

Definition

As used herein, the term “halogen” refers to F, Cl, Br and I.Preferably, halogen is selected from F, Cl, and Br.

As used herein, the term “alkyl” refers to straight-chain or branchedchain alkyl. Preferably, alkyl is C1-C4 alkyl, such as methyl, ethyl,propyl, iso-propyl, butyl, iso-butyl, tert-butyl and so on.

As used herein, the term “deuterated” means that one or more hydrogensof compounds or groups are substituted by deuteriums. “Deuterated” canbe mono-substituted, bi-substituted, multi-substituted ortotal-substituted. The terms “one or more deuterium-substituted” and“substituted by deuterium once or more times” can be usedinterchangeably.

In one embodiment, the deuterium content in a deuterium-substitutedposition is at least greater than the natural abundance of deuterium(0.015%), preferably >50%, more preferably >75%, more preferably >95%,more preferably >97%, more preferably >99%, more preferably >99.5%.

In one embodiment, the compound of formula (I) comprises at least onedeuterium atom, preferably three deuterium atoms, and more preferablyfive deuterium atoms.

As used herein, the term “compound CM4306” is4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-N-methylpicolinamide.

As used herein, the term “compound CM4307” is4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-N-(methyl-d₃)picolinamide.

As used herein, the term “compound CM4308” is4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluorophenoxy)-N-methylpicolinamide.

As used herein, the term “compound CM4309” is4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluorophenoxy)-N-(methyl-d₃)picolinamide.

As used herein, the term “TsOH” represents p-toluenesulfonic acid.Therefore, CM4307.TsOH represents the p-toluenesulfonate of CM4307.CM4309.TsOH represents the p-toluenesulfonate of CM4309.

Deuterium-Substituted ω-Diphenylurea

The preferred deuterated ω-diphenylurea compounds of the invention havethe structure of formula I:

wherein,

X is N or N⁺—O⁻;

R¹ is halogen (such as F, Cl or Br), one or more deuterium-substitutedor perdeuterated C1-C4 alkyl;

R² is non-deuterated C1-C4 alkyl, one or more deuterium-substituted orperdeuterated C1-C4 alkyl, or partly or totally halogen-substitutedC1-C4 alkyl;

each of R³, R⁴, R⁵, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ is independentlyhydrogen, deuterium, or halogen (such as F, Cl or Br);

R⁶ is hydrogen, deuterium, or one or more deuterium-substituted orperdeuterated C1-C4 alkyl;

R⁷ is hydrogen, deuterium, or one or more deuterium-substituted orperdeuterated C1-C4 alkyl; and

provided that at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹,R¹², R¹³ or R¹⁴ is deuterated or is deuterium.

In one embodiment, the deuterium content at a deuterium-substitutedposition is at least greater than the natural abundance of deuterium(0.015%), preferably >30%, more preferably >50%, more preferably >75%,or >95%, or >99%.

In one embodiment, except for H, all or almost all (>99wt %) of theelements (such as N, C, O, F, etc.) of compound (I) are naturallyexisting elements with highest abundance, such as ¹⁴N, ¹²C, ¹⁶O and ¹⁹F.

In one embodiment, compounds of formula (I) at least contain onedeuterium atom, preferably three deuterium atoms, and more preferablyfive deuterium atoms.

In one embodiment, R¹ is halogen, preferably chlorine.

In one embodiment, R² is trifluoromethyl.

In one embodiment, R⁶ or R⁷ is independently selected from hydrogen,deuterium, deuterated methyl, or deuterated ethyl; preferably,mono-deuterated methyl, bi-deuterated methyl, tri-deuterated methyl,mono-deuterated ethyl, bi-deuterated ethyl, tri-deuterated ethyl,tetra-deuterated ethyl, or penta-deuterated ethyl.

In one embodiment, R⁶ or R⁷ is independently selected from hydrogen,methyl or tri-deuterated methyl.

In one embodiment, R³, R⁴ or R⁵ is independently selected from hydrogenor deuterium.

In one embodiment, R⁸, R⁹, R¹⁰ or R¹¹ is independently selected fromhydrogen or deuterium.

In one embodiment, R¹², R¹³ or R¹⁴ is independently selected fromhydrogen or deuterium.

In one embodiment, said compounds are selected from:

N-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea(or4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)-phenoxy)-N-(methyl-d₃)picolinamide);

4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-2-(N-(methyl-d₃)aminoformyl)pyridine-1-oxide;

N-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(2,6-d₂-4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea;

N-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-d-6-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea;

orN-(4-chloro-3-(methyl-d₃)phenyl)-N′-(4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea.

or4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluoro-phenoxyl)-N-(methyl-d₃)picolinamide.

Intermediate

As used herein, the term “the intermediate of the invention” is2-(methyl-d₃)isoindoline-1,3-dione (or N-(methyl-d₃)benzosuccinimide);

Such intermediate can be called as “deuterated methyl phthalimide”.

Except for H, all or almost all (>99 wt %) of the elements (such as N,C, O, etc.) of the above compounds are naturally existing elements withhighest abundance, such as ¹⁴N, ¹²C and ¹⁶O.

Preparation

The preparation methods of the intermediates of the invention andcompound (I) are described in detail as below. However, these specificmethods are not provided for the limitation of the invention. Thecompounds of the invention can be readily prepared by optionallycombining any of the various methods described in the specification orvarious methods known in the art, and such combination can be readilycarried out by the skilled in the art.

The method for preparing un-deuterated ω-diphenylurea and thephysiologically compatible salts thereof used in the invention is known.The deuterated ω-diphenylurea can be prepared in the same route usingthe corresponding deuterated compounds as starting materials. Forexample, compound (I) can be prepared according to the method describedin WO2000/042012, except that the deuterated material is used instead ofun-deuterated material in the reaction.

In general, during the preparation, each reaction is conducted in aninert solvent, at a temperature between room temperature to refluxtemperature (such as 0-80° C., preferably 0-50° C.). Generally, thereaction time is 0.1-60 hours, preferably, 0.5-48 hours.

Taking CM4307 as an example, an optimized preparation route is shown asfollows:

Moreover, CM4309 can be prepared using the above route with3-fluoro-4-aminophenol instead of compound 4.

The deuterium can be introduced by using deuterated methylamine.

Deuterated methylamine or the hydrochloride thereof can be preparedthrough the following reactions. Deuterated nitromethane is obtained byreacting nitromethane with deuterium water in the presence of base (suchas sodium hydride, potassium hydride, deuterated sodium hydroxide,deuterated potassium hydroxide, potassium carbonate and the like) orphase-transfer catalyst. If necessary, the above experiment can berepeated to produce high-purity deuterated nitromethane. Deuteratednitromethane is reduced in the presence of zinc powder, magnesiumpowder, iron, or nickel and the like to form deuterated methylamine orthe hydrochloride thereof.

Furthermore, deuterated methylamine or the hydrochloride thereof can beobtained through the following reactions.

The key intermediate 3 can be synthesized from deuterated methanol(CD₃OD) through the following reactions.

The detailed preparation procedure is described in Example 1.

The main advantages of the present invention include:

(1) High-purity 2-(methyl-d₃)isoindoline-1,3-dione can be efficientlyprepared by the optimized process, which will be of benefit to thesubsequent reactions.

(2) Various of deuterated diphenylurea can be conveniently and highefficiently prepared by using the intermediate of the invention.

(3) The reaction conditions are milder and the operating process issafer.

The present invention will be further illustrated below with referencesto the specific examples. It should be understood that these examplesare only to illustrate the invention but not to limit the scope of theinvention. The experimental methods with no specific conditionsdescribed in the following examples are generally performed under theconventional conditions, or according to the manufacture's instructions.Unless indicated otherwise, parts and percentage are calculated byweight.

PREPARATION EXAMPLE 1 FOR THE INTERMEDIATE (1) Preparation of theintermediate 2-(methyl-d₃)isoindoline-1,3-dione

1. Preparation of methyl-d₃ p-toluenesulfonate

Sodium hydroxide (180 g, 4.5 mol, 5.0 eq) was added into water (288 mL).Methanol-d₃ (32.4 g, 900 mmol, 1.0 eq) was added at 0° C., and asolution of tosyl chloride (206 g, 1.1 mmol, 1.2 eq) in tetrahydrofuran(288 mL) was slowly added dropwise. The mixture was warmed to roomtemperature and stirred overnight. The mixture was neutralized bydropwise adding acetic acid (206 g) below 25° C. The reaction mixturewas filtered and separated. The aqueous phase was extracted with ethylacetate (100 mL). The filter cake was dissolved in water (300 mL) andextracted with ethyl acetate (200 mL). The organic phases were combined,washed with saturated sodium carbonate (100 mL) and saturated brine (100mL), dried over anhydrous sodium sulfate and filtered. The solvent inthe filtrate was removed under reduced pressure to afford the titlecompound (160.5 g, purity 99%, yield 94%) as a pale yellow liquid.

¹H NMR(CDCl₃, 400 MHz): δ63.20 (s, 3H), 7.71-7.75(m, 2H), 7.84-7.88(m,2H).

2. Preparation of 2-(methyl-d₃)isoindoline-1,3-dione

To N,N-dimethyl formamide (DMF, 225 mL), potassium phthalimide (166.7 g,0.9 mol, 2.0 eq) was added. Methyl-d₃ p-toluenesulfonate (85.2 g, 0.45mmol, 1.0 eq) prepared in the previous step was added dropwise at roomtemperature. The mixture was stirred at 60° C. for 0.5 hour, andfiltered immediately. The filter cake was washed with DMF (250 mL and100 mL) for two times. The DMF solutions were combined, and water (1150mL) was added dropwise at 0° C. to precipitate a white solid. The solidwas filtered and washed with water (100 mL×2). The obtained solid wasdried under vacuum to give the title compound (64 g, purity 99.6%, yield85%) as a white solid.

¹H NMR(CDCl₃, 400 MHz): δ7.71-7.77(m, 2H), 7.84-7.88(m, 2H).

3. Preparation of (methyl-d₃)amine hydrochloride

To a solution of distilled water (625 mL) and concentrated hydrochloricacid (625 mL, 7.5 mol, 15 eq), 2-(methyl-d₃)isoindoline-1,3-dione (82 g,0.5 mol, 1 eq) was added at room temperature. The mixture was heated to105° C. and refluxed overnight. The mixture was cooled to roomtemperature, filtered, and washed with distilled water (50 mL×2).Hydrochloric acid was removed under reduced pressure to afford a lightyellow solid. Anhydrous ethanol (140 mL) was added, and the resultedmixture was refluxed for 1 hour. The mixture was cooled to roomtemperature, and filtered. The solid was washed with ethanol (30 mL) anddried under vacuum to give the title compound (28 g, yield 80%) as awhite solid. ¹H NMR(DMSO-d₆, 400 MHz): δ8.05(br, 2H).

PREPARATION EXAMPLE 2 FOR THE INTERMEDIATE Prenaration of theintermediate 2-(methyl-d₃)isoindoline-1,3-dione

1: Preparation for 2-(methyl-d₃)isoindoline-1,3-dione

Step 2 in preparation example 1 was repeated except that: potassiumphthalimide was replaced by phthalimide (5.9 g, 40 mmol, 2.0 eq), andpotassium hydroxide (2.2 g, 40 mmol, 2.0 eq) was added in batches at 0°C. After stirred for 30 min, a solution of methyl-d₃ p-toluenesulfonate(3.8 g, 20 mmol, 1.0 eq) in DMF (10 mL) was added dropwise. After theaddition, the mixture was heated to 60° C. and stirred for 30 min.Conducting the filtration, washing and drying under vacuum by the samemethod as step 2 of preparation example 1 to give the title compound(2.1 g, purity 81%, yield about 62%) as a white solid.

PREPARATION EXAMPLE 3 FOR THE INTERMEDIATE

1: Preparation of 2-(methyl-d₃)isoindoline-1,3-dione

Step 2 in preparation example 1 was repeated except that: potassiumphthalimide is replaced by phthalimide (5.9 g, 40 mmol, 2.0 eq), andsodium hydride (80%, 1.2 g, 40 mmol, 2.0 eq) was added in batches at 0°C. After stirred for 30 min, a solution of methyl-d₃ p-toluenesulfonate(3.8 g, 20 mmol, 1.0 eq) in DMF (10 mL) was added dropwise. After theaddition, the mixture was heated to 60° C. and stirred for 30 min.Conducting the filtration, washing and drying in vacuum by the samemethod as step 2 of preparation example 1 to give the title compound(2.7 g, purity 86%, yield about 80%) as a white solid.

The above experiments showed that the new intermediate2-(methyl-d₃)isoindoline-1,3-dione could be prepared through the threemethods above. However, compared with the procedure by using phthalimideand potassium hydroxide or sodium hydride, using potassium phthalimideas an intermediate, on one hand, higher purity2-(methyl-d₃)isoindoline-1,3-dione could be obtained more efficiently,which was of benefit to the following reactions, on the other hand, thereaction condition is milder and the operation is safer because ofwithout using sodium hydride.

In Step 3 of preparation example 1 for the intermediate, because thehighly pure 2-(methyl-d₃)isoindoline-1,3-dione was used, high-purityN-(methyl-d₃)amine hydrochloride (purity>99.5%) was obtained.

EXAMPLE 1N-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-(N-(methyl-d3)aminoformyl)-4-pyridyloxy)phenyl)urea(Compound CM4307)

Route:

1. Preparation of 4-chloro-N-(methyl-d₃)picolinamide (3)

Into a 250 mL single-neck round-bottom flask equipped with waste gastreatment device, thionyl chloride (60 mL) was added. Anhydrous DMF (2mL) was dropwise added slowly while keeping temperature at 40-50° C.After the addition, the mixture was stirred for 10 min, and thennicotinic acid (20 g, 162.6 mmol) was added in portions in 20 min. Thecolor of the solution gradually changed from green into light purple.The reaction mixture was heated to 72° C., and refluxed for 16 hourswith agitation. A great amount of solid precipitate formed. The mixturewas cooled to room temperature, diluted with toluene (100 mL) andconcentrated to almost dry. The residue was diluted with toluene andconcentrated to dry. The residue was filtered and washed with toluene togive 4-chloropicolinoyl chloride as a light yellow solid. The solid wasslowly added into a saturated solution of (methyl-d₃)amine intetrahydrofuran in an ice-bath. The mixture was kept below 5° C. andstirred for 5 hours. The mixture was concentrated and ethyl acetate wasadded to give a white solid precipitate. The mixture was filtered, andthe filtrate was washed with saturated brine, dried over sodium sulfateand concentrated to give 4-chloro-N-(methyl-d₃)picolinamide (3) (20.68g, 73% yield) as a light yellow solid.

¹H NMR (CDCl₃, 300 MHz): 8.37 (d, 1H), 8.13 (s, 1H), 7.96(br, 1H),7.37(d, 1H).

2. Preparation of 4-(4-aminophenoxy)-N-(methyl-d₃)picolinamide (5)

To dry DMF (100 mL), 4-aminophenol (9.54 g, 0.087 mol) and potassiumtert-butoxide (10.3 g, 0.092 mol) were added in turn. The color of thesolution turned into deep brown. After stirring at room temperature for2 hours, 4-chloro-N-(methyl-d₃)picolinamide (3) (13.68 g, 0.079 mol) andanhydrous potassium carbonate (6.5 g, 0.0467 mol) were added. Thereaction mixture was heated to 80° C. and stirred overnight. TLCdetection showed the reaction was complete. The reaction mixture wascooled to room temperature, and poured into a solution of ethyl acetate(150 mL) and saturated brine (150 mL). The mixture was stirred and stoodfor separation. The aqueous phase was extracted with ethyl acetate(3×100 mL). The extracted layers were combined, washed with saturatedbrine (3×100 mL), dried over anhydrous sodium sulfate, and concentratedto afford 4-(4-aminophenoxy)-N-(methyl-d₃)picolinamide (18.00 g, 92%yield) as a light yellow solid.

¹H NMR (CDCl₃, 300 MHz): 8.32 (d, 1H), 7.99 (br, 1H), 7.66 (s, 1H),6.91-6.85 (m, 3H), 6.69 (m, 2H), 3.70 (br, s, 2H).

3. Preparation ofN-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea(CM4307)

To methylene chloride (120 mL), 4-chloro-3-trifluoromethyl-phenylamine(15.39 g, 78.69 mmol) and N,N′-carbonyldiimidazole (13.55 g, 83.6 mmol)were added. After stirred at room temperature for 16 hours, a solutionof 4-(4-aminophenoxy)-N-(methyl-d₃)picolinamide (18 g, 73 mmol) inmethylene chloride (180 mL) was slowly added dropwise and the mixturewas stirred at room temperature for another 18 hours. TLC detectionshowed the reaction was complete. The mixture was concentrated to about100 mL by removing methylene chloride through a rotary evaporator andstood for several hours at room temperature. A great amount of whitesolid precipitated. The mixture was filtered and the solid was washedwith abundant methylene chloride. The filtrate was concentrated byremoving part of solvent, and some solids precipitated again. Two partsof the solid were combined and washed with abundant methylene chlorideto affordN-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea(CM4307, 20.04 g, 58% yield) as a white powder (pure product).

¹H NMR (CD₃OD, 300 MHz): 8.48 (d, 1H), 8.00 (d, 1H), 7.55 (m, 5H), 7.12(d, 1H), 7.08 (s, 2H), ESI-HRMS m/z: C₂₁H₁₃D₃ClF₃N₄O₃, Calcd. 467.11,Found 490.07 (M+Na)⁺.

Furthermore, Compound CM4307 was dissolved in methylene chloride andreacted with m-chloroperoxybenzoic acid to afford the oxidizedderivative:4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl)ureido)phenoxy)-2-(N-(methyl-d₃)aminoformyl)pyridine-1-oxide.

EXAMPLE 2 Preparation of 4-chloro-N-(methyl-d₃)picolinamide (3)

a) Phthalimide (14.7 g, 0.1 mol), deuterated methanol (3.78 g, 0.105mol, 1.05 eq) and triphenylphosphine (28.8 g, 0.11 mol, 1.1 eq) weredissolved in anhydrous tetrahydrofuran. A solution of DEAD (1.1 eq) intetrahydrofuran was dropwise added under the ice-bath. After addition,the mixture was stirred for 1 hour at room temperature. The mixture waspurified by chromatography column, or the solvent in the mixture wasremoved, and then to the residue was added an appropriate amount of DCMand cooled in the refrigerator to precipitate the solid. The mixture wasfiltered and the filtrate was concentrated by a rotary evaporator, andthen the residue was purified by flash chromatography column to affordthe pure product of 2-(N-(methyl-d₃))-isoindole-1,3-dione (14.8 g, 90%yield).

Although the intermediate could be prepared through the above procedure,the purity of the product was low. It needed to be purified by columnchromatography to get the pure product. The work-ups were complicated,and the reagents (such as triphenylphosphine and diethylazodicarboxylate (DEAD), etc.) were environmentally unfriendly.Therefore, it was not suitable for industrial production.

b) 2-(N-(methyl-d₃))-isoindole-1,3-dione (12.5 g, 0.077 mol) wasdissolved in a suitable amount of hydrochloric acid (6 N, 50 mL) and themixture was refluxed for 24-30 hours in a sealed tube. The reactionmixture was cooled to room temperature and then cooled below 0° C. inthe refrigerator to precipitate the solid. The mixture was filtered andthe solid was washed with the cold deionized water. The filtrate wascollected and concentrated by a rotary evaporator to remove water. Theresulted solid was dried to afford (methyl-d₃)amine hydrochloride.Anhydrous DCM (100 mL) was added in (methyl-d₃)amine hydrochloride andmethyl 4-chloropicolinate hydrochloride (6.52 g, 0.038 mol, 0.5 eq) andsodium carbonate (12.2 g, 0.12 mol, 1.5 eq) were added. The reactionflask was sealed and placed in the refrigerator for one day. After TLCdetection showed the reaction was complete, the reaction mixture waswashed with water, dried, concentrated and purified by chromatographycolumn to afford 4-chloro-N-(methyl-d₃)picolinamide (compound (3), 5.67g, 86% yield). The structural feature of the compound was the same asthat in Example 1.

EXAMPLE 3 Preparation of diphenylurea compound CM4307 from2(N-(methyl-d₃))-isoindole-1,3-dione

1: Preparation of 4-chloro-N-(methyl-d₃)picolinamide A2

To a three-necked bottom flask with tetrahydrofuran (250 mL), methyl4-chloro-2-picolinate (50 g, 291 mmol, 1 eq) was added.N-(methyl-d₃)amine hydrochloride (31 g, 437 mmol, 1.5 eq) and anhydrouspotassium carbonate(400-mesh, 80 g, 583 mmol, 2 eq) were added withstirring. After the mixture was stirred at room temperature for 20 h,water (250 mL) and methyl tert-butyl ether (150 mL) were added. Themixture was stirred and separated. The aqueous layer was extracted withmethyl tert-butyl ether (100 mL). The organic phases were combined,dried over anhydrous sodium sulfate and filtered. The solvent in thefiltrate was removed under reduced pressure to obtain the product (48 g,purity 99%, yield 96%) as light yellow liquid.

¹H NMR(DMSO-d₆, 400 MHz): δ7.64(dd, J=2 Hz, 5.2 Hz, 1H), 7.97(d, J=1.6Hz, 1H), 8.54(d, J=5.2 Hz, 1H), 8.74(br, 1H).

MS (ESI, m/z) calcd. for C₇H₄D₃C1N₂O: 173, found : 174 [M+H]⁺

2: Preparation of 4-(4-aminophenoxy)-N-(methyl-d₃)picolinamide A3

Under nitrogen, 4-chloro-N-(methyl-d₃)picolinamide (2.4 g, 13.8 mmol, 1eq) was dissolved in dimethylsulfoxide (10 mL). To the resultedsolution, 4-aminophenyol (1.6 g, 14.9 mmol, 1.08 eq) was added followedby addition of potassium tert-butoxide (1.7 g, 15.2 mmol, 1.1 eq) inbatches. After the inner temperature was stable, the inner temperaturewas raised and kept at 80° C. for 4 hours. After the inner temperaturewas cooled to room temperature, iso-propanol (10 mL) was added withstirring to dilute the reaction mixture. Under nitrogen, concentratedhydrochloric acid (37%, 10 mL) was added dropwise, and the mixture wasstirred for 1 h, and then filtrated. The resulted solid was dissolved inwater (20 mL), activated carbon (0.1 g) was added. The resulted mixturewas stirred for 1 h and filtered. A solution of potassium carbonate (2 gpotassium carbonate dissolved in 6 mL water) was slowly added dropwiseinto the filtrate and some solids formed. The mixture was filtered, andthe solid was washed with water (5 mL), and dried in vacuum to give thetitle product (2.7 g, purity 99.9%, yield 78%) as a light brown crystal.

¹H NMR(DMSO-d₆, 400 MHz): δ5.19(br, 2H), 6.66-6.68(m, 2H), 6.86-6.88(m,2H), 7.07(dd, J=2.8 Hz, 5.6 Hz, 1H), 7.36(d, J=2.8 Hz, 1H), 8.45(d,J=5.6 Hz, 1H), 8.72(br, 1H).

MS (ESI, m/z) calcd. for C₁₃H₁₀D₃N₃O₂Cl: 246, found : 247[M+H]⁺

3. Preparation of4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-N-(methyl-d₃)picolinamide(CM4307)

Under nitrogen, 4-(4-aminophenoxy)-N-(methyl-d₃)picolinamide (2.6 g,10.6 mmol, 1 eq) was dissolved in dichloromethane (10 mL) anddimethylsulfoxide (3 mL). A solution of1-chloro-4-isocyanato-2-(trifluoromethyl)benzene (2.50 g, 11.1 mol, 1.05eq) in dichloromethane (8 mL) was slowly added dropwise to the abovesolution at room temperature. The resulted mixture was stirred for 20min. The mixture was cooled to 2° C. and water (10 mL) was addeddropwise. The resulted mixture was stirred for 0.5 h and filtered. Theproduct was washed with dichloromethane (10 mL). The resulted productwas dried in vacuum to give an off-white solid (4.7 g, purity 99.8%,yield 95.4%).

¹H NMR(DMSO-d₆, 400 MHz): δ7.15(dd, J=2.8 Hz, 5.6 Hz, 1H),7.17-7.19(m,2H), 7.40(d, J=2.4 Hz, 1H), 7.59-7.69(m, 4H), 8.13(d, J=2.4Hz, 1H), 8.51(d, J=6 Hz, 1H), 8.75(br, 1H), 8.90(br, 1H), 9.22(br, 1H).

MS (ESI, m/z) calcd. for C₂₁H₁₃D₃ClF₃N₄O₃: 467, found: 468[M +H]⁺

EXAMPLE 4 Preparation of4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-N-(methyl-d₃)picolinamidep-toluenesulfonate (CM4307.TsOH)

p-toluenesulfonic acid monohydrate (1.6 g, 8.5 mmol, 0.88 eq) wasdissolved in anhydrous ethanol (5 mL). The resulted solution wasfiltered, and then heated to 70° C. and kept at the same temperature.4-(4-3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-phenoxy)-2-(N-(methyl-d₃))picolinamide(4.5 g, 9.6 mol, 1 eq) and p-toluenesulfonic acid monohydrate (0.66 g,3.5 mmol, 0.36 eq) were suspended in ethanol (50 mL). The mixture washeated to 78° C. and refluxed for 40 min till the solid was fullydissolved. The resulted solution was slightly cooled and filtered whenthe solution was warm. The filtrate was heated till the innertemperature was 78° C. and refluxed for 1 h till the solution was clear.The pre-prepared ethanol solution ofp-toluenesulfonic acid monohydrateat 70° C. was quickly added in one time. Heating was stopped and themixture was cooled freely, stirred for 0.5 h at 0° C. and filtered. Theproduct was washed with anhydrous ethanol (5 mL×2), dried in vacuum at50° C. for 24 h to give a white to off-white solid (5.8 g, purity 99.3%,yield 93%).

¹H NMR(DMSO-d₆, 400 MHz): δ2.30(s, 3H), 7.15(d, J=8.8 Hz, 2H), 7.20(d,J=8.8 Hz, 2H), 7.23(dd, J=2.8 Hz, 6 Hz, 1H), 7.52(d, J=8 Hz, 2H),7.55(d, J=2.8 Hz, 1H), 7.63(d, J=8.8 Hz, 3H), 7.68(dd, J=2.4 Hz, 9.2 Hz,1H), 8.03(br, 1H), 8.14(d, J=2.4 Hz, 1H), 8.56(d, J=6 Hz, 1H), 8.91(br,1H), 9.17(br, 1H), 9.36(br, 1H).

¹³C NMR(DMSO-d₆, 400 MHz): δ21.1, 26.1, 111.7, 115.2, 117.0, 120.7(2C),121.6 (2C), 121.9, 122.8, 123.2, 124.6,125.6 (2C), 127.2, 129.0(2C),132.3, 138.8, 139.5, 139.9, 144.1, 146.6, 147.2, 152.8, 159.9, 170.7ppm.

Liquid chromatography condition: Agilent 1100 Series; chromatographiccolumn: Synergi 4μ POLAR-RP 80A, 250×4.6 mm, 4 μm; column temperature:25° C.; detection wavelength: UV 210 nm; mobile phase: A: ammoniumdihydrogen phosphate 10 mmol/L, B: methanol; injection volume: 10 μL;flow rate: 0.8 mL/min; run time: 70 min; gradient: 50% mobile phase Bfrom 0 to 15 min, mobile phase B being increased to 75% from 15 to 32min, then 75% mobile phase B eluting for 23 min from 32 to 55 min.retention time: 4.95 min (p-toluenesulfonic acid); 47.11 min (CM4307).

EXAMPLE 5 Preparation of Diphenylurea Compound CM4309 from2-(N-(methvl-d₃))-isoindole-1,3-dione

1: Preparation of 4-chloro-pyridyl-2-(N-(methyl-d₃))amide A2

The preparation method was identical to Example 3.

2: Preparation of 4-(4-amino-3-fluorophenoxy)-N-(methyl-d₃)picolinamideB1

Potassium tert-butoxide (15 g, 130 mmol, 1.3 eq) was suspended inN,N-dimethylacetylamide (DMA, 50 mL). A solution of4-amino-3-fluorophenol (16 g, 127 mmol, 1.3 eq) in DMA(50 mL) was slowlyadded dropwise at 0-5° C. The resulted mixture was stirred at roomtemperature for 20 min. The mixture was heated to 100° C. A solution of4-chloro-N-(methyl-d₃)picolinamide 3 (17 g, 97 mmol, 1 eq) in DMA (50mL) was slowly added dropwise. After the addition, the mixture wasstirred for 0.5 h, then cooled to room temperature, diluted with ethylacetate (1.5 L) and stirred for 0.5 h. Then the mixture was filtered toremove the inorganic salts. The filtrate was washed with water (500mL×3), dried over anhydrous sodium sulfate and concentrated underreduced pressure to remove the solvents. To the resulted crude productwas added ethanol (100 mL). The resulted mixture was refluxed for 2 h,then cooled to room temperature and filtered to give a brown solid (20g, HPLC purity 96%, yield 80%).

¹H NMR(CD₃OD, 400 MHz): δ6.74-6.77(m, 1H), 6.87(dd, J=2.4 Hz, 11.6 Hz,1H), 6.93(t, J=10 Hz, 1H), 7.02(dd, J=2.8 Hz, 6 Hz, 1H), 7.54(d, J=2.4Hz, 1H), 8.44(d, J=6 Hz, 1H). MS (ESI, m/z) calcd. for C₁₃H₉D₃ClN₃O₂:264, found : 265 [M+H]⁺

3. Preparation of4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluoro-phenoxy)-N-(methyl-d₃)picolinamide(CM4309)

At room temperature, 1-chloro-4-isocyanato-2-(trifluoromethyl)benzene(13 g, 58 mmol, 1.1 eq) was added into a single-necked bottom flask withdichloromethane (70 mL). A solution of4-(4-amino-3-fluorophenoxy)-N-(methyl-d₃)picolinamide (14 g, 53 mmol, 1eq) in dichloromethane (350 mL) was slowly added dropwise to the abovesolution. The resulted mixture was stirred at room temperature for 20 h.After the reaction was complete, the mixture was filtered and the solidwas washed with dichloromethane (20 mL×2) to obtain a light brown solid(13 g, purity 98%, yield 50%).

¹H NMR(DMSO-d₆, 400 MHz): δ 7.06-7.10(m, 1H), 7.19(dd, J=2.4 Hz, 5.6 Hz,1H), 7.35(dd, J=2.8 Hz, 12 Hz, 1H), 7.43(d, J=2.4 Hz, 1H), 7.63(m, 2H),8.14(br, 1H), 8.17(t, J=8.8 Hz, 1H), 8.53(d, J=5.6 Hz, 1H), 8.75(d,J=1.6 Hz, 1H), 8.78(br, 1H), 9.54(br, 1H).

MS (ESI, m/z) calcd. for C₂₁H₁₂D₃ClF₄N₄O₃: 485, found: 486 [M+H]⁺

EXAMPLE 6 Preparation of4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyOureido)-3-fluorophenoxy)-N-(methyl-d₃)picolinamidep-toluenesulfonate (CM4309.TsOH)

4-(4-(3-(4-chloro-3-(trifluoromethyl)phenyl]ureido)-3-fluorophenoxy)-N-(methyl-d₃)picolinamide(2.16 g, 4.33 mmol, 1 eq) was suspended in ethanol (50 mL), and themixture was refluxed till the solution was clear. The mixture wasfiltered when it was hot to remove the undissolved materials. Thefiltrate was refluxed till it was clear, and then p-toluenesulfonic acid(70%, 8.66 mmol, 1 mL, 2 eq) was quickly added in one time. The clearsolution was kept for about 0.5 min. The oil bath was turned off and thesolution was cooled to room temperature freely. The mixture was stirredfor 1 h and filtered. The resulted undissolved materials were dried invacuum at 50° C. for 20 h to give a white crystal (2.36 g, yield 83%).

¹H NMR(DMSO-d₆, 400 MHz): δ2.30(s, 3H), 7.09-7.14(m, 3H), 7.24(dd, J=2.4Hz, 5.2 Hz, 1H), 7.37(dd, J=2.4 Hz, 11.6 Hz, 1H), 7.48-7.50(m, 3H),7.61-7.66(m, 2H), 8.15-8.19(m, 2H), 8.56(d, J=5.6 Hz, 1H), 8.79(br, 1H),8.85(br, 1H), 9.56(br, 1H), 10.38(br, 1H).

Melting point: 240.7° C.-241° C.

EXAMPLE 7 Preparation ofN-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(2,6-d₂-4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea

The preparation method was identical with example 1, except that thetitle compound was obtained using 3,5-d₂-4-aminophenol instead ofp-aminophenol.

EXAMPLE 8 Preparation ofN-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-d-6-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea

The preparation method was identical with example 1, except that thetitle compound was obtained using 2-d-6-carboxylpyridine instead ofnicotinic acid.

EXAMPLE 9 Preparation ofN-(4-chloro-3-(methyl-d₃)phenyl)-N′-(4-(2-(N-(methyl-d₃)aminoformyl)-4-pyridyloxy)phenyl)urea

The preparation method was identical with example 1, except that thetitle compound was obtained using 5-amino-2-chloro-(methyl-d₃)-benzeneinstead of 5-amino-2-chloro-trifluorobenzene.

EXAMPLE 10 Pharmacokinetic Evaluation of Deuterated DiphenylureaCompounds in Rats

8 male Sprague-Dawley rats, 7-8 weeks-old and body weight about 210 g,were divided into two groups, 4 in each group (rat No.: control groupwas 13-16; experimental group was 9-12). The rats were orallyadministrated at a single dose of 3 mg/kg of (a) the undeuteratedcompoundN-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-(N-methyl-aminoformyl)-4-pyridyloxy)phenyl)urea(control compound CM4306) or (b)N-(4-chloro-3-(trifluoromethyl)phenyl)-N′-(4-(2-(N-(methyl-d₃)-aminoformyl)-4-pyridyloxy)phenyl)urea(Compound CM4307 of the invention) prepared in Example 1. Thepharmacokinetic differences of CM4306 and CM4307 were compared.

The rats were fed with the standard feed, given water andchlordiazepoxide. Chlordiazepoxide was stopped at the last night beforeexperiment, and given again two hours after the administration of thecompound. The rats were fasted for 16 hours before the test. Thecompound was dissolved in 30% PEG400. The time for collecting orbitalblood was 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours afteradministrating the compound.

The rats were anaesthetized briefly by inhaling ether. A 300 μL orbitalblood sample was collected into the tubes containing a 30 μL 1% heparinsaline solution. The tubes were dried overnight at 60° C. before beingused. After the blood samples were sequentially collected, the rats wereanaesthetized by ether and sacrificed.

After the blood samples were collected, the tubes were gently reversedat least five times immediately to mix the contents sufficiently, andplaced on the ice. The blood samples were centrifuged at 4° C. at 5000rpm for 5 minutes to separate the serum and red blood cells. 100 μLserum was removed to a clean plastic centrifugal tube by pipettor, andthe name of the compound and time point was labeled on the tube. Serumwas stored at -80° C. before LC-MS analysis.

The results were shown in FIGS. 1-2. The results showed that, comparedwith CM4306, the half-life (T_(1/2)) of CM4307 was longer [11.3±2.1hours for CM4307 and 8.6±1.4 hours for CM4306, respectively], area underthe curve (AUC_(0∞)) of CM4307 was significantly increased [11255±2472ng·h/mL for CM4307 and 7328±336 ng·h/mL for CM4306, respectively], andapparent clearance of CM4307 was reduced [275±52 mL/h/kg for CM4307 and410±18.7 mL/h/kg for CM4306, respectively].

The above results showed that, the compound of the present invention hadbetter pharmacokinetics properties in the animal, and thus had betterpharmacodynamics and therapeutic effects.

In addition, the metabolism for the compound of the present invention inorganism was changed through deuteration. In particular, thehydroxylation of phenyl became more difficult, which led to thereduction of first-pass effect. In such cases, the dose can be changed,long-acting preparations can be formed, and the applicability can beimproved by using long-acting preparations.

Furthermore, the pharmacokinetics was also changed through deuteration.Since another hydrate film is fully formed by deuterated compounds, thedistribution of deuterated compounds in organisms is significantlydifferent from that of the non-deuterated compounds.

EXAMPLE 11 The Pharmacodynamics Evaluation of CM4307 for InhibitingTumor Growth of Human Hepatocellular Carcinoma SMMC-7721 in Nude MiceXenograft Model

70 Balb/c nu/nu nude mice, 6 weeks-old, female, were bought fromShanghai Experimental Animal Resource Center (Shanghai B&K UniversalGroup Limited).

SMMC-7721 cells were commercially available from Shanghai Institutes forBiological Science, CAS (Shanghai, China).

The establishment of tumor nude mice xenograft model: SMMC-7721 cells inlogarithmic growth period were obtained. After cell number counting, thecells were suspended in 1× PBS, and the concentration of the cellsuspension was adjusted to 1.5×10⁷/ml. The tumor cells were inoculatedunder the skin of right armpit of nude mice with a 1 ml syringe,3×10⁶/0.2 ml/mice. 70 nude mice were inoculated in total.

When the tumor size reached 30-130 mm³, 58 mice were divided randomlyinto different groups. The difference of the mean value of tumor in eachgroup was less than 10%, and drugs were started to be administrated.

The test doses for each groun were listed in the following table.

Ani- Com- Adminis- Dose Group mal pounds tration (mg/kg) Method 1 10control po 0.1 ml/ qd × 2 weeks (solvent) 10 g BW 2 8 CM4306 po 10 mg/kgqd × 2 weeks 3 8 CM4306 po 30 mg/kg qd × 2 weeks 4 8 CM4306 po 100mg/kg  qd × 2 weeks 5 8 CM4307 po 10 mg/kg qd × 2 weeks 6 8 CM4307 po 30mg/kg qd × 2 weeks 7 8 CM4307 po 100 mg/kg  qd × 2 weeks

Animal body weight and tumor size were tested twice a week during theexperiment. Clinical symptoms were recorded every day. At the end of theadministration, the tumor size was recorded by taking pictures. Onemouse was sacrificed in each group and tumor tissue was taken and fixedin 4% paraformaldehyde. Observation was continued after theadministration, and when the mean size of tumor was larger than 2000mm³, or the dying status appeared, the animals were sacrificed, grossanatomy was conducted, and the tumor tissue was taken and fixed in 4%paraformaldehyde.

The formula for calculating the tumor volume (TV) is: TV=a×b²/2, whereina, b independently represent the length and the breadth of the tumor.The formula for calculating the relative tumor volume (RTV) is:RTV=Vt/V₀, wherein V₀ is the tumor volume at the beginning of theadministration, and Vt is the tumor weight when measured. The index forevaluating the antitumor activity is relative tumor increment rate T/C(%), and the formula is: T/C (%)=(T_(RTV)/C_(RTV))×100%, wherein,T_(RTV) is the RTV of the treatment group, and C_(RTV) is the RTV of thenegative control group.

Evaluation standard for efficacy: it is effective if the relative tumorincrement rate T/C (%) is <40% and p<0.05 by statistics analysis.

The results were shown in FIG. 3. CM4306 and CM4307 were intragastricadministrated every day for 2 weeks at doses of 10, 30, 100 mg/kg, andboth compounds showed the dose-dependent effect of the inhibition oftumor growth. At the end of administration, T/C % of CM4306 was 56.9%,40.6% and 32.2%, respectively. T/C % of CM4307 was 53.6%, 40.8% and19.6%. T/C % for 100 mg/kg dose groups was <40%, and tumor volume wassignificantly different (p<0.01) from the control group, indicating thesignificant effect in inhibiting tumor growth.

Compared with CM4306, the inhibitory efficacy of tumor growth at dosing100 mg/kg of CM4307 was stronger (the T/C % for CM4307 and CM4306 is19.6% and 32.2%, respectively, at day 15), there was significantdifference in tumor volume between groups (p<0.01). Compared withCM4306, the absolute value of tumor inhibition rate for CM4307 increasedmore than 10%, the relative value increased about 60%(32.2%/19.6%−1=64%), and CM4307 showed more significant effect forinhibiting tumor growth.

In addition, during the experiment, no other drug-relevant toxic effectswere observed.

EXAMPLE 12

The Activity for Inhibiting c-Kit, PDGFR-β Protein Tyrosine Kinase atthe Molecular Level

1. Method

The activity of diphenylurea compounds for inhibiting c-Kit, PDGFR-βprotein tyrosine kinase on molecular level was tested by Enzyme-LinkedImmunosorbent Assay (ELISA).

Compounds to be tested: CM4306, CM4308 and CM4309.

Main Reagents:

Reaction substrate Poly(Glu,Tyr)_(4:1) was bought from Sigma. Monoclonalantibody against tyrosine phosphate PY99 was bought from Santa Cruz.Horseradish peroxidase labeled sheep-against-rat IgG was bought fromCalbiochem; ATP, DTT, OPD were bought from Amresco; microplates werebought from Corning; Su1 1248 was bought from Merk.

The Testing Method:

See Roskoski, R., Jr. Sunitinib: a VEGF and PDGF receptor protein kinaseand angiogenesis inhibitor. Biochem Biophys Res Commun, 356: 323-328,2007. The details comprise:

Kinase reaction substrate Poly(Glu,Tyr)_(4:1) was diluted to 20 μg/ml bypotassium-free PBS, and the microplates were coated. Diphenylureasamples to be tested were added in the wells of the coated microplates(the samples to be tested were made from the stock solution 10⁻² M inDMSO, and diluted to the desired concentration with reaction bufferbefore use. the resulted solution was added into the wells to get thefinal concentration of 10⁻⁵ mol/L in 100 μL reaction system). At thesame time, the positive control compound Su11248 was added into thepositive control wells.

ATP solution diluted with reaction buffer (the final concentration ofATP is 5 μM) followed by addition of tyrosine kinase diluted withreaction buffer. The total volume of reaction system was 100 μL. At thesame time, the negative control wells and the control wells withoutenzyme were set up.

The reaction system was placed in a wet box, and shaked for 1 h at 37°C. shielding from light. After the reaction was completed, the plateswere washed with T-PBS for three times. Antibody PY99 was added (100μL/well) , and the plates were shaked for 30 min at 37° C. After thereaction was completed, the plates were washed with T-PBS for threetimes. Horseradish peroxidase labeled sheep-against-rat IgG was added(100 μL/well), and the plates were shaked for 30 min at 37° C. After thereaction was complete, the plates were washed with T-PBS for threetimes. OPD developing liquid was added (100 μL/well), and reacted for1-10 min at room temperature shielding from light. 50 μL of 2 M H₂SO₄was added to quench the reaction. A₄₉₂ value was determined usingwavelength-adjustable microplate reader VERSAmax.

The inhibition ratio of samples can be calculated by the followingformula:

${{inhibition}\mspace{14mu} {ratio}\mspace{14mu} \%} = {\left( {1 - \frac{\begin{matrix}{{{OD}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {compound}} -} \\{{OD}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {control}\mspace{14mu} {without}\mspace{14mu} {enzyme}}\end{matrix}}{\begin{matrix}{{{OD}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {negative}\mspace{14mu} {control}} -} \\{{OD}\mspace{14mu} {value}\mspace{14mu} {of}\mspace{14mu} {control}\mspace{14mu} {without}\mspace{14mu} {enzyme}}\end{matrix}}} \right) \times 100\%}$

2. Results

Compound % Inhibition ratio for tyrosine kinase at 10 μM No. c-Kit meanvalue PDGFR-β mean value results CM4306 80.6 85.8 effective CM4308 84.087.1 effective CM4309 85.2 88.2 effective Su11248 90.2 93.9 effective(positive drug)

The above results are the mean values for experiments in duplicate.

3. The Evaluation Standard and the Results

The compound has efficacy if the inhibition rate of the tested compoundat the experimental concentration 10⁻⁵ mol/L was greater than 50%. Thecompound has no efficacy if the inhibition rate was less than 50%,provided that the inhibitory activity of the positive control compoundmeets the reference range.

The results showed that the inhibition ratios of CM4306, CM4308, CM4309against protein tyrosine kinases c-Kit, PDGFR-β were more than 50%.Therefore, they had significantly inhibitory activity against c-Kit,PDGFR-β tyrosine kinase at the molecular level.

All literatures mentioned in the present application are incorporated byreference herein, as though individually incorporated by reference.Additionally, it should be understood that after reading the aboveteaching, many variations and modifications may be made by the skilledin the art, and these equivalents also fall within the scope as definedby the appended claims.

1-10. (canceled)
 11. A method for preparing2-(methyl-d₃)isoindoline-1,3-dione, wherein, the method comprises: (a)in an inert solvent, reacting a alkali metal salt of phthalimide withcompound A,

wherein, Z is CH₃, O-CD₃ or

wherein R is methyl, nitro or halogen, to form2-(methyl-d₃)isoindoline-1,3-dione:


12. The method according to claim 11, wherein, said inert solventincludes N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA),dimethylsulfoxide (DMSO), N-methylpyrrolidone (NMP), tetrahydrofuran,1,4-dioxane, or the combination thereof
 13. The method according toclaim 11, wherein, in step (a), the reaction temperature is −10° C. toreflux temperature.
 14. The method according to claim 11, wherein, instep (a), said alkali metal salt of phthalimide includes potassiumphthalimide, sodium phthalimide, lithium phthalimide, or the combinationthereof.
 15. The method according to claim 11, wherein, in step (a),said compound A includes methyl-d₃ 4-methyl-benzenesulfonate, methyl-d₃3-nitrobenzenesulfonate, or methyl-d₃ 4-nitrobenzenesulfonate.
 16. Themethod according to claim 11, wherein, prior to step (a) of said method,it further includes step (a1): under a basic condition and in an inertsolvent, reacting deuterated methanol with tosyl chloride to formmethyl-d₃ 4-methyl-benzenesulfonate.
 17. A method for preparing a saltof (methyl-d₃)amine, wherein, the method comprises: reacting2-(methyl-d₃)isoindoline-1,3-dione with hydrochloride acid to form(methyl-d₃)amine hydrochloride:


18. The method according to claim 17, wherein reacting2-(methyl-d₃)isoindoline-1,3-dione with 6N hydrochloride acid:


19. An intermediate wherein, said intermediate is2-(methyl-d₃)isoindoline-1,3-dione:


20. A process for preparing a compound having one of the followingformula:

comprising reacting the compound of claim 19 with an appropriateprecursor to form the specified compound.